In order to maintain normal hemostasis, a delicate balance between activators of fibrinolysis, such as tissue plasminogen activator (tPA) and urokinase (uPA), and inhibitors of fibrinolysis, such as .alpha..sub.2 -antiplasmin, .alpha..sub.2 -macroglobulin, and plasminogen activator inhibitor-1 (PAI-1), must be achieved. Stump et al., Semin. Thromb. Hemos., Vol. 16, No. 3. pp. 260-273 (1990); Lijnen et al., Fibrinolysis, Vol. 3, pp. 67-77 (1989). Much work has been carried out in an attempt to understand the precise biological role and balance of each of these activators and inhibitors in various physiological and pathophysiological states, and to fashion therapeutics useful in achieving or maintaining the normal hemostatic state. Despite this intensive effort, a great deal still remains to be understood.
Some researchers in this field have focused on understanding the role of the protein plasminogen activator inhibitor-1 (PAI-1). PAI-1 is believed to be the principal physiological regulator of tPA, serving as a specific, fast-acting inhibitor of the serine protease tPA, as well as an inhibitor of uPA. It has been found that PAI-1 is a protein of a molecular weight of about 50,000 which binds to tPA in a 1:1 complex and inactivates it. Van Mourik et al., J. Biol. Chem., Vol. 259, pp. 14914-14921 (1984); Colucci et al., J. Clin. Invest., Vol. 75, pp. 818-824 (1985); Almer et al., Thromb. Research, Vol. 47, pp. 335-339 (1987). Several clinical studies suggest that elevated levels of PAI-1 may contribute to the pathogenesis of various thrombotic disorders, including myocardial infarction, deep vein thrombosis, and disseminated intravascular coagulation, by acting to reduce the net endogenous fibrinolytic capacity. Hamsten et al., New England J. of Medicine, Vol. 313, pp. 1557-1563 (1985); Wiman et al , J. Lab. Clin. Med., Vol 105, pp. 265-270 (1985). In another more recent study, however, it was found that induction of endogenous PAI-1 protein failed to alter thrombolysis caused by an infusion of tPA. Colucci et al., J. Clin. Invest., Vol. 78, pp. 138-144 (1986). The latter experiments suggest that administration of exogenous PAI-1 would not alter fibrinolysis in vivo. Indeed, it has been reported that exogenously administered PAI-1 has no effect on primary bleeding. Further, there is evidence that PAI-1 actually serves to inhibit thrombin, an action which would serve to increase bleeding, if anything. Ehrlich et al., J. Biol. Chem., Vol. 265, pp. 13029-13035 (1990).
Recently, a recombinant form of plasminogen activator inhibitor-1 was produced in active form from E. coli. Reilly et al., J. Biol. Chem., Vol. 265, No. 16, pp. 9570-9574 (1990); Davis et al., U.S. Ser. No. 350,264, filed May 11, 1989, entitled "High Level Expression of Functional Human Plasminogen Activator Inhibitor in E. coli". This rPAI-1 protein was found to inhibit tPA and uPA with a second order rate constant in the range of 2-5.times.10.sup.-7 M.sup.-1 s.sup.-1. The use of this recombinant PAI-1 protein as a therapeutic agent to counteract excessive or inappropriate fibrinolysis was first suggested in the patent application Davis et al., U.S. Ser. No. 350,264, filed May 11, 1989, entitled "High Level Expression of Functional Human Plasminogen Activator Inhibitor in E. coli". Further studies have suggested that this recombinant PAI-1 protein, when administered intravenously, is more potent than the antifibrinolytic agent .epsilon.-amino caproic acid (EACA) in reversing tPA-induced hemorrhage in a rabbit model Racanelli et al., Fibr., Vol. 4, Suppl. 3, p. 43 (1990). Another recombinant PAI-1 protein purified in an inactive form and activated by treatment with guanidinium hydrochloride was found to reverse tPA-induced bleeding in aspirin-treated rabbits when administered systemically by injection. Vaughan et al., J. Clin. Invest , Vol. 84, pp. 586-591 (1989).
Hemostatic agents suitable for topical administration (rather than oral administration) to control localized bleeding are advantageous in that they are capable of being directly targeted to the area of interest. This potentially allows a more rapid therapeutic response, using lower dosage amounts, while at the same time minimizing the likelihood of side effects characteristic of some systemically administered hemostatic agents. Topically applied hemostatic agents which have been used during surgery to control localized blood loss from primary bleeding have included thrombin, fibrin glue, gelatin sponge, microfibrillar collagen, and oxidized cellulose. Schwartz, Principles of Surgery, Ch. 3, 5th ed., New York, McGraw-Hill (1984).
Several other agents have also been tested topically for hemostatic use. For example, in one study the anti- fibrinolytic agent EACA was administered locally, systemically, and both locally and systemically, in hemophiliacs undergoing dental extractions and Factor VIII therapy. Stajcic et al., Int. J. Oral Surg., Vol. 14, No. 4, pp. 339-345 (1985). The authors found the therapeutic effects to be unsatisfactory where EACA was only locally applied. Id. at pp. 339 & 340. Later in Allingham et al., Arch. Ophthalmol., Vol. 105, No. 10, pp. 1421-1423 (1988), EACA was employed topically in a rabbit to treat traumatic hyphema, a unique condition of ocular bleeding. In certain applications the antifibrinolytic agent aprotinin has also been applied topically to control bleeding, such as in neurosurgery where aprotinin was employed in combination with artificial fibrin glues. Verstraete, Drugs, Vol. 29, pp 236-261 (1985).
Although some progress has been made in this area, new and/or better agents for topical administration to control localized blood loss are needed. The present invention, which is directed to the first to successful application of the inhibitor PAI-1 topically to control localized bleeding, is directed to this important end.